NATURAL RESOURCE LETTER

Two new flavonoids from Centella asiatica (Linn.)

Ravi Subban Æ A. Veerakumar Æ R. Manimaran ÆK. M. Hashim Æ Indira Balachandran

Received: 12 October 2007 / Accepted: 20 December 2007 / Published online: 13 February 2008

� The Japanese Society of Pharmacognosy and Springer 2008

Abstract Two new flavonoids named castilliferol 1 and

castillicetin 2, as well as a known compound, isochloro-

genic acid 3, were isolated from the whole plant of

Centella asiatica. Isolates 1 and 2 exhibited good anti-

oxidant activity using 2,2-diphenyl-1-picryl hydrazyl

radical solution with IC50 values of 23.10 and 13.30,

respectively. The structures of these isolates were deter-

mined by analytical and spectral data, including 1-D and 2-

D NMR spectra.

Keywords Apiaceae � Centella asiatica � Castilliferol �Castillicetin � Isochlorogenic acid

Introduction

Centella or Indian pennywort, Centella asiatica (Linn.)

Urban syn. Hydrocotyle asiatica Linn., belonging to the

family Apiaceae, is valued in Indian systems of medicine

for improving memory and for the treatment of nervine

disorders and skin diseases [1]. It has been used extensively

as a memory enhancer. The herb is known as Brahmi in

Unani medicine, Mandookaparni in Ayurveda and Gotu

Kola in the Western world. In India, the plant was previ-

ously confused with Bacopa monnieri Wettst., as both were

sold in the market under the name ‘‘Brahmi.’’ However,

this controversy has been resolved and it is has been con-

cluded that Brahmi is B. monnieri and Mandookaparni is

C. asiatica [2]. The plant is indigenous to South-East Asia,

India, Sri Lanka, parts of China, the western South Sea

Islands, Madagascar, South Africa, South-East USA,

Mexico, Venezuela, Columbia and eastern South America.

Previously, triterpenoid acids [2–8], volatile and fatty oils

[2, 9], alkaloids [9], glycosides [2, 3, 9–13], flavonoids

[2, 11], and steroids [2, 9] have been isolated from the

different parts of the plant. This paper deals with the iso-

lation and structural elucidation of two new constituents

(1, 2) and one known constituent 3, and the antioxidant

effect of these isolates.

Results and discussion

Castilliferol 1 was isolated as a yellow powder which

decomposes at 274 �C. The molecular formula C24H16O8

was determined from elemental analysis and from the

pseudomolecular ion peak in the ESIMS negative mode

([M-H]- m/z 431) and the ESIMS positive mode ([M+H]+

m/z 433) spectra. The IR spectrum showed absorption

bands at 3,344 (OH), 1,680, 1,664 (conjugated carbonyl),

1,517 cm-1 (Ar ring). The UV spectrum displayed bands at

260 and 366 nm and showed that compound 1 was a fla-

vone derivative. The band at 366 nm was shifted by

+44 nm upon the addition of NaOMe and by +33 nm when

NaOAc was added, indicative of a free hydroxyl group at

the C-40 position. A bathochromic shift of 45 nm in the

band I absorption maximum with AlCl3 and AlCl3/HCl

revealed the presence of a chelated hydroxyl in C-5. The13C NMR spectrum of 1 (Table 1) displayed signals for 24

carbons, which were edited by HSQC and DEPT into 12

methines and 12 quaternary carbons consisting of two

R. Subban (&) � K. M. Hashim � I. Balachandran

Phytochemistry Division, Centre for Medicinal Plants Research,

Arya Vaidya Sala, Kottakkal, Kerala, India 676503

e-mail: ravisubban@rediffmail.com

A. Veerakumar � R. Manimaran

JSS College of Pharmacy, Udhagamandalam,

Nilgiris, Tamilnadu, India 643001

123

J Nat Med (2008) 62:369–373

DOI 10.1007/s11418-008-0229-0

carbonyls at dc 172.03 and 174.30 and seven oxygenated

carbons. The 1H NMR spectrum (500 MHz, CH3COCH3–

d6) showed resonances due to four hydroxyl groups at d11.5 (2H), 12.6 (1H) and 13.2 (1H) and two pairs of

AA0BB0-type signals; one pair at d 7.95 (d, J = 8.0 Hz)

and 6.90 (d, J = 8.0 Hz) belonged to the protons at the 20,60 and 30,50 protons of the flavonoid moiety and was par-

tially overlapped with the alkene protons of the p-

coumaroyl system. The other pair at d 8.18 (d, J = 8 Hz)

and 7.00 (d, J = 8 Hz) belongs to the 200,600 and 300,500

positions of the p-coumaroyl system. Due to the vicinal

coupling constants in the signals at d 7.95(d, J = 8.0 Hz,

H-700) and d 6.90 (d, J = 8.0 Hz, H-800) being 8.00 Hz,

they were assigned to a cis-configured double bond. For a

trans-configured double bond the coupling constant value

will be on the order J = 15.0 Hz [14, 15]. A cross peak

was observed in the NOSEY spectrum between the above

two signals, supporting the cis configuration. The two

doublets at d 6.40 and 6.52 (each 1H, J = 2 Hz) were

assigned to H-8 and H-6, respectively, of the flavonoid

nucleus. The carbon resonance signals at d 170.6, 161.6,

148.4, 132.6, 133.1, 115.7, 115.6 and 115.4 in the 13C

NMR spectrum supported the presence of p-coumaroyl

residue. A cleavage at the carbonyl carbon of the

p-coumaroyl moiety was observed in the mass spectrum by

exhibiting a signal at m/z 286 of the aglycone moiety. The

HMBC NMR spectrum showed key correlations between

H-700(d 7.95) and C-200, C-600 and C-900 (d 132.6 and 131.6,

170.6, respectively), H-200, H-600 (d 8.18) and C-400

(d 161.6), H-20, H-60 (d 7.95) and C-40 (d 148.8), and

between H-30, H-50 (d 6.90) and C-10 (d 125.6). Without the

coumaroyl moiety, it was similar to that of kaempferol. The

structure of castilliferol 1 was therefore elucidated as

kaempferol-3-p-coumarate.

Castillicetin 2 was isolated as a yellow powder which

decomposes at 310 �C. Elemental analysis and ESIMS

negative mode indicated the molecular formula to be

C24H16O10. The UV spectrum has adsorptions at 253, 329

and 368 nm, showing that compound 2 was a flavone

derivative. The band at 368 nm was shifted by +46 nm

upon the addition of NaOMe and by +31 nm when NaOAc

was added, indicative of a free hydroxyl group at the C-40

position. A bathochromic shift of 32 nm in the band at

253 nm with AlCl3 and AlCl3/HCl revealed the presence of

a chelated hydroxyl in C-5. The 13C NMR spectrum of 2

displayed signals for 24 carbons, which were edited by

Table 1 NMR data for

compounds 1 and 2Position 1 2

dH (mult., J [HZ]) COSY dC dH (mult., J [HZ]) COSY dC

2 157.8 159.2

3 136.5 136.4

4 172.0 171.7

5 162.7 158.4

6 6.52 (d, 2.1) H-8 99.4 6.32 (d, 2.1) H-8 99.6

7 164.4 158.6

8 6.40 (d, 2.1) H-6 94.6 6.50 (d, 2.1) H-6 94.4

9 148.2 157.8

10 104.4 104.7

10 125.6 125.5

20 7.95 (d, 8.0) H-30 130.7 7.12 (d, 2.1) H-60 115.3

30 6.90 (d, 8.0) H-20 116.1 144.8

40 148.8 148.5

50 6.90 (d, 8.0) H-60 116.0 7.00 (d, 8.0) H-60 116.5

60 7.95 (d, 8.0) H-50 130.6 7.75 (dd, 8.0, 2.1) H-50,20 117.7

10 0 133.1 126.4

20 0 8.18 (d, 8.0) H-30 0 132.6 7.00 (d, 2.1) H-60 0 117.4

30 0 7.00 (d, 8.0) H-20 0 115.7 148.6

40 0 161.6 152.3

50 0 7.00 (d, 8.0) H-60 0 115.6 6.80 (d, 8.0) H-60 0 119.5

60 0 8.18 (d, 8.0) H-50 0 131.6 7.85 (dd, 8.0, 2.1) H-50 0,20 0 117.1

70 0 7.95 (d, 8.0) H-80 0 148.4 7.65 (d, 8.3) H-80 0 148.6

80 0 6.90 (d, 8.0) H-70 0 115.4 6.98 (d, 8.3) H-70 0 116.6

90 0 170.6 170.5

370 J Nat Med (2008) 62:369–373

123

HSQC. SEFT NMR spectra of 2 showed the presence of 10

methine and 14 quaternary carbons, which includes two

carbonyls at d 171.7 and 173.4 and nine oxygenated car-

bons. The 1H NMR spectrum (500 MHz, CH3COCH3–d6)

showed resonances due to six hydroxyl groups at d 12.4

(4H) and 11.8 (2H) and a pair of ABX proton spin systems

was observed at d 7.12 (d, J = 2.1 Hz), 7.00 (dd, J = 8.0,

2.1 Hz), 7.75 (dd, J = 8.0, 2.1 Hz) and 7.00 (dd, J = 8.0,

2.1 Hz), 6.80 (d, J = 8.0 Hz), and 7.85 (dd, J = 8.0,

2.1 Hz), characteristic of two 1,3,4-trisubstituted benzene

units. In addition, two doublet signals at d 6.32 and 6.50

(each 1H, J = 2 Hz), assignable to H-8 and H-6, respec-

tively, of the flavonoid nucleus, were also observed. Two

doublets at d7.65 and 6.98 (each 1H, J = 8.3 Hz) appeared

as an AB system. Due to the vicinal coupling constant

being 8.3 Hz, they were assigned to a cis-configured dou-

ble bond. A cross peak was observed in the NOSEY

spectrum between the above two signals supporting the cis

configuration.

The carbon signals at 170.5, 159.3, 152.6, 148.6, 126.4,

119.5, 117.4, 117.1 and 116.6 in the 13C NMR spectrum

indicated the presence of a caffeoyl moiety. Without the

caffeoyl moiety signals, it was similar to that of Quercetin.

The HMBC NMR spectrum showed key correlations

between H-200 (d 7.00) and C-700 (d 148.6), H-200 (d 7.00)

and C-400 (d 152.3), H-600 (d 7.85) and C-700 (d 148.6), H-600

(d 7.85) and C-400 (d 152.3), H-700 (d 7.65) and C-200

(d 117.4), H-700 (d 7.65) and C-600 (d 117.1), H-500 (d 6.80)

and C-300 (d 148.62), H-700 (d 7.65) and C-900 (d 170.52),

H-20 (d 7.12) and C-40 (d 148.50), H-20 (d 7.12) and C-2

(d 159.2), H-60 (d 7.75) and C-2 (d 159.2). Detailed anal-

ysis of the 1-D, 2-D NMR data enabled the full assignment

of all protons and carbons of this compound (Table 1). The

structure of castillicetin 2 was therefore elucidated as

Quercetin-3-caffeate.

Compound 3 was identified as isochlorogenic acid by

comparing its spectral data with those in the literature

[16, 17].

Antioxidant activity was measured by a decrease in the

absorbance at 516 nm of methanolic solution of colored

2,2-diphenyl-1-picryl hydrazyl radical solution (DPPH)

brought about by the sample. A stock solution of DPPH

(1.3 mg/ml methanol) was prepared such that 75 ll of it in

3 ml methanol gave an initial absorbance of 0.9. This stock

solution was used to measure the antioxidant activity.

Decreases in the absorbance in the presence of compound 1

and 2 at different concentrations were noted after 20 min.

IC50 was calculated from percentage of inhibition

(Table 2). Gallic acid was used as positive control. Cas-

tilliferol 1 and castillicetin 2 exhibited good antioxidant

activity with IC50 values of 23.10 and 13.31 lg/ml,

respectively, when compared to the control (IC50 value of

14.58 lg/ml) (Fig. 1).

Materials and methods

General experimental procedures

Melting points were determined on a Veego (Mumbai,

India) melting point apparatus and are uncorrected. IR

spectra were recorded as KBr pellets on a PerkinElmer

(Waltham, MA, USA) 2000 FTIR spectrophotometer. UV

spectra were measured on a Shimadzu (Tokyo, Japan) UV-

2101 PC UV–Vis scanning spectrophotometer. 1H NMR

(500 MHz) and 13C NMR (150.9 MHz) spectra were

measured on a JEOL (Tokyo, Japan) JNM LA-500

instrument using acetone-d6 solvent. ESI-MS were deter-

mined on an Agilent (Palo Alto, CA, USA) MSD 1100

single-quadrapole spectrometer. Elemental analysis was

carried out on a PerkinElmer 240C instrument. For TLC,

precoated Si gel 60 F254 plates were used and spots were

Table 2 Antioxidant activity of compounds 1 and 2

Sample Concentration

(lg/ml)

Percentage of

inhibition

IC50

(lg/ml)

1 5 9.04 23.10

25 57.08

50 84.74

2 5 18.10 13.31

25 75.44

50 85.24

Gallic

acid

14.58

OHO

OH

OH O

O C

CH CH

OH

O

OH

OH

CO

COOH

C

OH

HO

HO OH

HO

O

O OH

O

12

3

45

6

1'

2'

3'

4'5'

6'

1''

2''

3''4''

5''

6''

7''

ß

aß'

a'

OHO

OH O

O C

CH CH

OH

O

OH

8

9

10

2

35

6

1'

1''

2'

2'' 3''5'

5''

6'

6''7''8''

9''

3'

4'

4''

1

2

3

Fig. 1 Structures of compounds 1, 2 and 3

J Nat Med (2008) 62:369–373 371

123

detected under UV light and further visualized by spraying

with vanillin-sulfuric acid. Column chromatography was

carried out on Si gel (60–120 mesh, Merck, Darmstadt,

Germany). DPPH was purchased from Sigma Chemical

Co. (St. Louis, MO, USA). HPLC purification was per-

formed on a LC-8A, SPD with FRC-10A Shimadzu

apparatus.

Plant material

The whole plant material of C. asiatica was collected from

the Nilgris district, Tamilnadu, India in August 2005. The

Botany Department of Government Arts College,

Udhagamandalam, The Nilgiris identified the plant

material and a voucher specimen has been deposited at the

college.

Extraction and isolation

The air-dried material (2 kg, whole plant) was ground and

extracted exhaustively with MeOH–H2O (6:4, 3 9 5.0 l) for

2 h. The extracts were combined and freed of solvent to give

160 g of residue. The extract was dissolved in a mixture of

MeOH–H2O (1:1, 500 ml) and extracted with n-butanol

(3 9 300 ml) to give, after concentration in vacuo, 102 g of

a n-butanol soluble extract. The residue was subjected to

column chromatography on 200 g of Si gel and eluted with

EtOAc (500 ml) followed with EtOAc containing increasing

amounts of MeOH. Eighty fractions (25 ml each) were

collected and pooled according to their similiarity on

analytical TLC plates and dried. Combined fractions 5–38

(CAI, 10.7 g), which were eluted with EtOAc, and 52–79

(CAII, 9 g), eluted with EtOAc–MeOH (9:1), were obtained.

Fraction CAI was subjected to chromatography on 140 g of

Si gel and eluted with pet. ether to start with and further

fractions were obtained by increasing the polarity of the

solvent by suitable additions of EtOAc and MeOH. Sixty

fractions (25 ml each) were collected and pooled according

to their similarity on analytical TLC plates and concentrated

in vacuo. This resulted in fractions 22–39 (A, 14 g)

and fractions 42–60 (B, 220 mg) when eluted with

EtOAc-pet.ether (3:7). A was rechromatographed in another

Si gel column, eluted with CH3COCH3-pet. ether (3:7) to

yield 1 (500 mg). B was rechromatographed in another Si gel

column, eluted with CH3COCH3-pet. ether (3:7) to yield 2

(109 mg). Compounds 1 and 2 were purified by RP-HPLC

(Inertsil C-18 column, 2.50 9 4.6 mm, acetonitrile and

orthophosphoric acid buffer, 1.5 ml/min, photodiode array

detection monitored at 254 nm). CAII was subjected to

column chromatography on 55 g diaion and eluted with

MeOH–H2O (9:1) to yield 3 (41 mg).

Kaempferol-3-p-coumarate 1: yellow powder; mp 274

�C (dec.); UV (MeOH) kmax (log e) 366 (3.51), 260 (2.52)

nm; (MeOH + NaOMe) kmax (log e) 410 (3.48), 320

(1.90), 272 (2.41) nm; (MeOH + NaOAc) kmax (log e) 399

(3.49), 320 (1.87), 271 (2.43) nm; (MeOH + AlCl3) kmax

(log e) 411 (3.29), 320 (1.98), 302 (1.69), 272 (2.51) nm;

(MeOH + AlCl3 + HCl) kmax (log e) 411 (3.31), 3.20

(1.89), 301 (1.68), 271 (2.48) nm; IR (KBr) mmax 3,344,

1,694, 1,664, 1,517, 1,267 cm-1; 1H NMR (CH3COCH3–

d6, 500 MHz) and 13C NMR , see Table 1; elemental

analysis, found: C 66.6532, H 3.7327 (calc. C 66.6536, H

3.7319 for C24H16O8 ); ESI-MS negative mode m/z 431

[M-H]- ; ESI-MS positive mode m/z 433 [M+H]+.

Quercetin-3-caffeate 2: yellow powder; mp 310 �C

(dec.); UV (MeOH) kmax (log e) 253 (2.46), 329 (1.87), 363

(3.21) nm; (MeOH + NaOCH3) kmax (log e) 269 (2.36),

320 (1.71), 344 (1.98), 409 (3.42); (MeOH + NaOAc)

kmax (log e) 273 (2.35), 320 (1.92), 394 (3.12);

(MeOH + AlCl3) kmax (log e) 273 (2.49), 308 (1.67), 332

(1.89), 433 (3.22); (MeOH + AlCl3 + HCl) 273 (2.49),

307 (1.59), 340 (1.91), 401 (3.17); IR (KBr).1H NMR (CH3COCH3–d6, 500 MHz) and 13C NMR,

see Table 1; elemental analysis, found: C 62.0578, H

3.4753 (calc. C 62.0590, H 3.4747, for C24H16O10); ESI-

MS negative mode m/z 463 [M-H]-; ESI-MS positive

mode m/z 465 [M+H]+.

Antioxidant activity

Different concentrations (5, 25, 50 lg/ml) of test solution

and reference standard were placed in different microtitre

plates; 10 ll DPPH (1.3 mg/ml in methanol) solution was

added to each well. Finally, sufficient methanol was added

to make the volume up to 250 ll in each well. The reaction

mixture was mixed and incubated at room temperature for

20 min; the absorbance was measured at 516 nm. Gallic

acid was used as positive control.

References

1. Shakir Jamil S, Qudsia N, Mehboobus S (2007) Centella asiatica(Linn.) urban—a review. Nat Prod Radiance 6(2):158–170

2. CSIR (1992) The wealth of India: a dictionary of Indian raw

materials and industrial products (Raw Materials series). Publi-

cations and Information Directorate, CSIR, New Delhi. Rev Ser

3:428–430

3. Singh B, Rastogi RP (1969) A reinvestigation of the triterpenes of

Centella asiatica. Phytochemistry 8:917

4. Asolkar LV, Kakkar KK, Chakre OJ (1992) Second supplement

to glossary of Indian medicinal plants with active principles.

Publications and Information Directorate, CSIR, New Delhi, Part

I, pp 189–190

5. Schaneberg BT, Mikell JR, Bedir E Khan IA (2003) An improved

HPLC method for quantitative determination of six triterpenes in

372 J Nat Med (2008) 62:369–373

123

Centella asiatica extracts and commercial products. Pharmazie

58(6):381–384

6. Rastogi RP, Mehrotra BN (1993) Compendium of Indian

medicinal plants, vol I (1960–1969). Central Drug Institute,

Lucknow, India and Publication and Information Directorate,

CSIR, Dr. KS Krishnan Marg, New Delhi, India, pp 96

7. Rastogi RP, Sarkar B, Dhar ML (1960) Chemical examination of

Centella asiatica Linn. Isolation of the chemical constituents.

J Sci Ind Res Sect B 19:252

8. Rastogi RP, Mehrotra BN (1993) Compendium of Indian

medicinal plants, vol II (1970–1979). Central Drug Institute,

Lucknow, India and Publication and Information Directorate,

CSIR, Dr. KS Krishnan Marg, New Delhi, India, pp 169–170

9. Chopra RN, Nayar SL, Chopra IC (1956) Glossory of Indian

medicinal plants. Council for Scientific and Industrial Research,

New Delhi, p 58

10. Agarwal VS (1990) Economic plants of India. Kailash Prakashan,

Calcutta, p 137

11. Rastogi RP, Mehrotra BN (1993) Compendium of Indian

medicinal plants, vol III (1980–1984). Central Drug Institute,

Lucknow, India and Publication and Information Directorate,

CSIR, Dr. KS Krishnan Marg, New Delhi, India, pp 159

12. Chopra RN, Chopra IC, Varma BS (1992) Supplement to glos-

sary of Indian medicinal plants. CSIR, New Delhi, p 14

13. Datta T, Basu UP (1962) Triterpenoids. Thankuniside and

thankunic acid: a new triterpene glycoside and acid from Centellaasiatica Linn(Urb.). J Sci Ind Res Sect B 21:239

14. Liu J, Feng Z, Xu J, Wang Y, Zhang P (2007) Rare biscoumarins

and a chlorogenic acid derivative from Erycibe obtusifolia.

Phytochemistry 68(13):1775–1780

15. Kruthiventi AK, Krishnaswamy NR (2000) Constituents of the

flowers of Persea gratissima. Fitoterapia 71(1):94–96

16. Peng LY, Mei SX, Jiang B, Zhou H, Sun HD (2000) Constituents

from Lonicera Japonica. Fitoterapia 71(6):713–715

17. Corse J, Ludin RE, Waiss Jr AC (1965) Identification of several

components of isochlorogenic acid. Phytochemistry 4(3):527–529

J Nat Med (2008) 62:369–373 373

123